Nocardia rubra cell-wall skeleton mitigates whole abdominal irradiation-induced intestinal injury via regulating macrophage function

Abstract

Background: Ionizing radiation (IR)-induced intestinal injury is a major side effect and dose-limiting toxicity in patients receiving radiotherapy. There is an urgent need to identify an effective and safe radioprotectant to reduce radiation-induced intestinal injury. Immunoregulation is considered an effective strategy against IR-induced injury. The purpose of this article was to investigate the protective effect of Nocardia rubra cell wall skeleton (Nr-CWS), an immunomodulator, on radiation-induced intestinal damage and to explore its potential mechanism. Methods: C57BL/6 J male mice exposed to 12 Gy whole abdominal irradiation (WAI) were examined for survival rate, morphology and function of the intestine and spleen, as well as the gut microbiota, to comprehensively evaluate the therapeutic effects of Nr-CWS on radiation-induced intestinal and splenetic injury. To further elucidate the underlying mechanisms of Nr-CWS-mediated intestinal protection, macrophages were depleted by clodronate liposomes to determine whether Nr-CWS-induced radioprotection is macrophage dependent, and the function of peritoneal macrophages stimulated by Nr-CWS was detected in vitro. Results: Our data showed that Nr-CWS promoted the recovery of intestinal barrier function, enhanced leucine-rich repeat-containing G protein-coupled receptor 5+ intestinal stem cell survival and the regeneration of intestinal epithelial cells, maintained intestinal flora homeostasis, protected spleen morphology and function, and improved the outcome of mice exposed to 12 Gy WAI. Mechanistic studies indicated that Nr-CWS recruited macrophages to reduce WAI-induced intestinal damage. Moreover, macrophage depletion by clodronate liposomes blocked Nr-CWS-induced radioprotection. In vitro, we found that Nr-CWS activated the nuclear factor kappa-B signaling pathway and promoted the phagocytosis and migration ability of peritoneal macrophages. Conclusions: Our study suggests the therapeutic effect of Nr-CWS on radiation-induced intestinal injury, and provides possible therapeutic strategy and potential preventive and therapeutic drugs to alleviate it.

Keywords

Nocardia rubra cell wall skeleton / Ionizing radiation / Radiation-induced intestinal injury / Intestinal flora / Radiation protection / Peritoneal macrophages

References

[1] Schaue D, McBride WH. Opportunities and challenges of radiotherapy for treating cancer. Nat Rev Clin Oncol. 2015; 12: 527-40.

[2] Barnett GC, West CM, Dunning AM, Elliott RM, Coles CE, Pharoah PD, et al. Normal tissue reactions to radiotherapy: towards tailoring treatment dose by genotype. Nat Rev Cancer. 2009; 9: 134-42.

[3] Hauer-Jensen M, Wang J, Boerma M, Fu Q, Denham JW. Radiation damage to the gastrointestinal tract: mechanisms, diagnosis, and management. Curr Opin Support Palliat Care. 2007; 1: 23-9.

[4] Kiang JG, Olabisi AO. Radiation: a poly-traumatic hit leading to multi-organ injury. Cell Biosci. 2019; 9: 25.

[5] Macia IGM, Lucas Calduch A, Lopez EC. Radiobiology of the acute radiation syndrome. Rep Pract Oncol Radiother. 2011; 16: 123-30.

[6] Kumagai T, Rahman F, Smith AM. The microbiome and radiation induced-bowel injury: evidence for potential mechanistic role in disease pathogenesis. Nutrients. 2018; 10: 1405. https://doi.org/10.3390/nu10101405.

[7] Gorbunov NV, Sharma P. Protracted oxidative alterations in the mechanism of hematopoietic acute radiation syndrome. Antioxidants (Basel). 2015; 4: 134-52.

[8] Shadad AK, Sullivan FJ, Martin JD, Egan LJ. Gastrointestinal radiation injury: symptoms, risk factors and mechanisms. World J Gastroenterol. 2013; 19: 185-98.

[9] Lopez M, Martin M. Medical management of the acute radiation syndrome. Rep Pract Oncol Radiother. 2011; 16: 138-46.

[10] Shadad AK, Sullivan FJ, Martin JD, Egan LJ. Gastrointestinal radiation injury: prevention and treatment. World J Gastroenterol. 2013; 19: 199-208.

[11] Lacave-Lapalun JV, Benderitter M, Linard C. Flagellin or lipopolysaccharide treatment modified macrophage populations after colorectal radiation of rats. J Pharmacol Exp Ther. 2013; 346: 75-85.

[12] Tajima G, Delisle AJ, Hoang K, O’Leary FM, Ikeda K, Hanschen M, et al. Immune system phenotyping of radiation and radiation combined injury in outbred mice. Radiat Res. 2013; 179: 101-12.

[13] Smith PD, Smythies LE, Shen R, Greenwell-Wild T, Gliozzi M, Wahl SM. Intestinal macrophages and response to microbial encroachment. Mucosal Immunol. 2011; 4: 31-42.

[14] Tanaka M, Abe S. Different activities of antitumor immunomodulators to induce neutrophil adherence response. Drug Discov Ther. 2019; 13: 299-305.

[15] Hine AM, Loke P. Intestinal macrophages in resolving inflammation. J Immunol. 2019; 203: 593-9.

[16] Chiaranunt P, Tai SL, Ngai L, Mortha A. Beyond immunity: underappreciated functions of intestinal macrophages. Front Immunol. 2021; 12: 749708.

[17] Meziani L, Deutsch E, Mondini M. Macrophages in radiation injury: a new therapeutic target. Oncoimmunology. 2018; 7: e1494488.

[18] Yi J, Chen C, Liu X, Kang Q, Hao L, Huang J, et al. Radioprotection of EGCG based on immunoregulatory effect and antioxidant activity against (60)Cogamma radiation-induced injury in mice. Food Chem Toxicol. 2020; 135: 111051.

[19] Nadella V, Ranjan R, Senthilkumaran B, Qadri S, Pothani S, Singh AK, et al. Podophyllotoxin and Rutin modulate M1 (iNOS+) macrophages and mitigate lethal radiation (LR) induced inflammatory responses in mice. Front Immunol. 2019; 10: 106. https://doi.org/10.106.10.3389/fimmu.2019.00106.

[20] Venkateswaran K, Shrivastava A, Agrawala PK, Prasad AK, Devi SC, Manda K, et al. Mitigation of radiation-induced gastrointestinal injury by the polyphenolic acetate 7,8-diacetoxy-4-methylthiocoumarin in mice. Sci Rep. 2019; 9: 14134.

[21] Izumi S, Ogawa T, Miyauchi M, Fujie K, Okuhara M, Kohsaka M. Antitumor effect of Nocardia rubra cell wall skeleton on syngeneically transplanted P388 tumors. Cancer Res. 1991; 51: 4038-44.

[22] Azuma I, Taniyama T, Yamawaki M, Sugimura K, Yamamura Y. Adjuvant and antitumor activities of Nocardia cell-wall skeletons. Gan. 1976; 67: 733-6.

[23] Chen W, Zhang Y, Zhao C, Shao S, Zhang Y, Li X, et al. Nocardia Rubra Cell Wall skeleton up-regulates T cell subsets and inhibits PD-1/PD-L1 pathway to promote local immune status of patients with high-risk human papillomavirus infection and cervical intraepithelial neoplasia. Front Immunol. 2020; 11: 612547.

[24] Zhao J, Du HJ, Liao QP. Effect of Nocardia rubra cell wall skeleton on the growth of HeLa cell line infected with HPV. Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi. 2007; 21: 20-2.

[25] Zhao J, Zhan SB, Li XQ, Zhou L, Yang YJ, Liao QP. Effect of Nocardia rubra cell wall skeleton (Nr-CWS) on oncogenicity of TC-1 cells and anti-human papillomavirus effect of Nr-CWS in lower genital tract of women. Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi. 2007; 21: 340-2.

[26] Sakatani M, Ogura T, Masuno T, Kishimoto S, Yamamura Y. Effect of Nocardia rubra cell wall skeleton on augmentation of cytotoxicity function in human pleural macrophages. Cancer Immunol Immunother. 1987; 25: 119-25.

[27] Meng Y, Sun J, Wang X, Ma Y, Kong C, Zhang G, et al. The biological macromolecule Nocardia rubra cell-wall skeleton as an avenue for cell-based immunotherapy. J Cell Physiol. 2019; 234: 15342-56.

[28] Tao Y, Wang G, Zhai J, Zhang N. Functional modulation of CD8+ T cell by approved novel immune enhancer: Nocardia rubra Cell-Wall skeletons (Nr-CWS). Int Immunopharmacol. 2020; 78: 106023. https://doi.org/10.1016/j.intimp.2019.106023.

[29] Wang G, Wu J, Miao M, Dou H, Nan N, Shi M, et al. Nocardia rubra cell-wall skeleton promotes CD4(+) T cell activation and drives Th1 immune response. Int J Biol Macromol. 2017; 101: 398-407.

[30] Zhou Q, Shen B, Huang R, Liu H, Zhang W, Song M, et al. Bacteroides fragilis strain ZY-312 promotes intestinal barrier integrity via upregulating the STAT3 pathway in a radiation-induced intestinal injury mouse model. Front Nutr. 2022; 9: 1063699. https://doi.org/10.3389/fnut.2022.1063699.

[31] Guo H, Chou WC, Lai Y, Liang K, Tam JW, Brickey WJ, et al. Multi-omics analyses of radiation survivors identify radioprotective microbes and metabolites. Science. 2020; 370. https://doi.org/10.1126/science.aay9097.

[32] Li M, Gu Y, Ma YC, Shang ZF, Wang C, Liu FJ, et al. Kruppel-like factor 5 promotes epithelial proliferation and DNA damage repair in the intestine of irradiated mice. Int J Biol Sci. 2015; 11: 1458-68.

[33] Bensemmane L, Squiban C, Demarquay C, Mathieu N, Benderitter M, Le Guen B, et al. The stromal vascular fraction mitigates radiation-induced gastrointestinal syndrome in mice. Stem Cell Res Ther. 2021; 12: 309.

[34] Forstner JF. Intestinal mucins in health and disease. Digestion. 1978; 17: 234-63.

[35] Rogakou EP, Pilch DR, Orr AH, Ivanova VS, Bonner WM. DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139. J Biol Chem. 1998; 273: 5858-68.

[36] Pan MR, Peng G, Hung WC, Lin SY. Monoubiquitination of H2AX protein regulates DNA damage response signaling. J Biol Chem. 2011; 286: 28599-607.

[37] Yan KS, Chia LA, Li X, Ootani A, Su J, Lee JY, et al. The intestinal stem cell markers Bmi1 and Lgr5 identify two functionally distinct populations. Proc Natl Acad Sci U S A. 2012; 109: 466-71.

[38] Gehart H, Clevers H. Tales from the crypt: new insights into intestinal stem cells. Nat Rev Gastroenterol Hepatol. 2019; 16: 19-34.

[39] Bevins CL, Salzman NH. Paneth cells, antimicrobial peptides and maintenance of intestinal homeostasis. Nat Rev Microbiol. 2011; 9: 356-68.

[40] Gerassy-Vainberg S, Blatt A, Danin-Poleg Y, Gershovich K, Sabo E, Nevelsky A, et al. Radiation induces proinflammatory dysbiosis: transmission of inflammatory susceptibility by host cytokine induction. Gut. 2018; 67: 97-107.

[41] Zhou K. Strategies to promote abundance of Akkermansia muciniphila, an emerging probiotics in the gut, evidence from dietary intervention studies. J Funct Foods. 2017; 33: 194-201.

[42] Chen W, Liu F, Ling Z, Tong X, Xiang C. Human intestinal lumen and mucosa-associated microbiota in patients with colorectal cancer. PLoS One. 2012; 7: e39743. https://doi.org/10.1371/journal.pone.0039743.

[43] Zhang S, Wang H, Liu Y, Tao T, Zeng Z, Zhou Y, et al. Nocardia rubra cell-wall skeleton influences the development of cervical carcinoma by promoting the antitumor effect of macrophages and dendritic cells. Cancer Med. 2022; 11: 1249-68.

[44] Hu K, Xu Y, Li X, Du P, Lu Y, Lyu G. The Nocardia Rubra Cell Wall skeleton regulates macrophages and promotes wound healing. Curr Issues Mol Biol. 2022; 44: 5995-6005.

[45] Wang J, Zhao B, Sun L, Jiang L, Li Q, Jin P. Smart thermosensitive poloxamer hydrogels loaded with Nr-CWs for the treatment of diabetic wounds. PLoS One. 2022; 17: e0279727. https://doi.org/10.1371/journal.pone.0279727.

[46] Hauer-Jensen M, Denham JW, Andreyev HJ. Radiation enteropathy-pathogenesis, treatment and prevention. Nat Rev Gastroenterol Hepatol. 2014; 11: 470-9.

[47] Chou DB, Frismantas V, Milton Y, David R, Pop-Damkov P, Ferguson D, et al. Author correction: on-chip recapitulation of clinical bone marrow toxicities and patient-specific pathophysiology. Nat Biomed Eng. 2020; 4: 477.

[48] Zhang C, Zeng W, Yao Y, Xu B, Wei X, Wang L, et al. Naringenin ameliorates radiation-induced lung injury by lowering IL-1beta level. J Pharmacol Exp Ther. 2018; 366: 341-8.

[49] Zheng Y, Pang X, Zhu X, Meng Z, Chen X, Zhang J, et al. Lycium barbarum mitigates radiation injury via regulation of the immune function, gut microbiota, and related metabolites. Biomed Pharmacother. 2021; 139: 111654.

[50] de Reijke TM, de Boer EC, Schamhart DH, Kurth KH. Immunostimulation in the urinary bladder by local application of Nocardia rubra cell wall skeleton preparation (Rubratin) for superficial bladder cancer immunotherapy-a phase I/II study. Urol Res. 1997; 25: 117-20.

[51] Wang Y, Hu Y, Ma B, Wang F, Liu S, Xu J, et al. Nocardia rubra cell wall skeleton accelerates cutaneous wound healing by enhancing macrophage activation and angiogenesis. J Int Med Res. 2018; 46: 2398-409.

[52] Jian YP, Yang G, Zhang LH, Liang JY, Zhou HL, Wang YS, et al. Lactobacillus plantarum alleviates irradiation-induced intestinal injury by activation of FXR-FGF15 signaling in intestinal epithelia. J Cell Physiol. 2022; 237: 1845-56.

[53] Mothersill C, Seymour C. Radiation-induced bystander effects: past history and future directions. Radiat Res. 2001; 155: 759-67.

[54] Parsons Jr WB, Watkins CH, Pease GL, Childs Jr DS. Changes in sternal marrow following roentgen-ray therapy to the spleen in chronic granulocytic leukemia. Cancer. 1954; 7: 179-89.

[55] Allaire JM, Crowley SM, Law HT, Chang SY, Ko HJ, Vallance BA. The intestinal epithelium: central coordinator of mucosal immunity. Trends Immunol. 2018; 39: 677-96.

[56] Sato T, Sase M, Ishikawa S, Kajita M, Asano J, Sato T, et al. Characterization of radioresistant epithelial stem cell heterogeneity in the damaged mouse intestine. Sci Rep. 2020; 10: 8308.

[57] Lu L, Jiang M, Zhu C, He J, Fan S. Amelioration of whole abdominal irradiation-induced intestinal injury in mice with 3,3’-Diindolylmethane (DIM). Free Radic Biol Med. 2019; 130: 244-55.

[58] Rooks MG, Garrett WS. Gut microbiota, metabolites and host immunity. Nat Rev Immunol. 2016; 16: 341-52.

[59] Cui M, Xiao H, Li Y, Zhou L, Zhao S, Luo D, et al. Faecal microbiota transplantation protects against radiation-induced toxicity. EMBO Mol Med. 2017; 9: 448-61.

[60] Li Y, Dong J, Xiao H, Zhang S, Wang B, Cui M, et al. Gut commensal derived-valeric acid protects against radiation injuries. Gut Microbes. 2020; 11: 789-806.

[61] Gu J, Zhao L, Chen YZ, Guo YX, Sun Y, Guo Q, et al. Preventive effect of sanguinarine on intestinal injury in mice exposed to whole abdominal irradiation. Biomed Pharmacother. 2022; 146: 112496.

[62] Viola MF, Boeckxstaens G. Niche-specific functional heterogeneity of intestinal resident macrophages. Gut. 2021; 70: 1383-95.

[63] Bain CC, Schridde A, Schridde A. Origin, differentiation, and function of intestinal macrophages. Front Immunol. 2018; 9: 02733.

[64] Na YR, Stakenborg M, Seok SH, Matteoli G. Macrophages in intestinal inflammation and resolution: a potential therapeutic target in IBD. Nat Rev Gastroenterol Hepatol. 2019; 16: 531-43.

[65] Mukherjee D, Coates PJ, Lorimore SA, Wright EG. Responses to ionizing radiation mediated by inflammatory mechanisms. J Pathol. 2014; 232: 289-99.

[66] Frontiers Editorial O. Retraction: Podophyllotoxin and Rutin modulate M1 (iNOS+) macrophages and mitigate lethal radiation (LR) induced inflammatory responses in mice. Front Immunol. 2022; 13: 10613.

[67] Riehl TE, Alvarado D, Ee X, Zuckerman A, Foster L, Kapoor V, et al. Lactobacillus rhamnosus GG protects the intestinal epithelium from radiation injury through release of lipoteichoic acid, macrophage activation and the migration of mesenchymal stem cells. Gut. 2019; 68: 1003-13.

[68] Yang Y, Li L, Xu C, Wang Y, Wang Z, Chen M, et al. Cross-talk between the gut microbiota and monocyte-like macrophages mediates an inflammatory response to promote colitis-associated tumourigenesis. Gut. 2020; 70: 1495-506.

[69] Bain CC, Bravo-Blas A, Scott CL, Perdiguero EG, Geissmann F, Henri S, et al. Constant replenishment from circulating monocytes maintains the macrophage pool in the intestine of adult mice. Nat Immunol. 2014; 15: 929-37.

[70] Mortha A, Chudnovskiy A, Hashimoto D, Bogunovic M, Spencer SP, Belkaid Y, et al. Microbiota-dependent crosstalk between macrophages and ILC3 promotes intestinal homeostasis. Science. 2014; 343: 1249288.

[71] Wu T, Li H, Su C, Xu F, Yang G, Sun K, et al. Microbiota-derived short-chain fatty acids promote LAMTOR2-mediated immune responses in macrophages. mSystems. 2020; 5: e00587-20. https://doi.org/10.1128/mSystems.00587-20.

[72] Earley AM, Graves CL, Shiau CE. Critical role for a subset of intestinal macrophages in shaping gut microbiota in adult zebrafish. Cell Rep. 2018; 25: 424-36.

[73] Baldwin Jr AS. The NF-kappa B and I kappa B proteins: new discoveries and insights. Annu Rev Immunol. 1996; 14: 649-81.

[74] Burdelya LG, Krivokrysenko VI, Tallant TC, Strom E, Gleiberman AS, Gupta D, et al. An agonist of toll-like receptor 5 has radioprotective activity in mouse and primate models. Science. 2008; 320: 226-30.